The first step of metastasis involves the attachment of cancer cells to tissues around the primary site, i.e., to the extracellular matrix (ECM) via cell surface integrins and other adhesion receptors. Integrin targets of the ECM include fibronectin, fibrinogen, vitronectin, collagen and laminin. Integrins mediate cell-cell and cell-substratum interactions and are involved in bidirectional signaling that links the ECM with cytoskeletal proteins. In the second step, cancer cells secrete digestive enzymes that degrade the surrounding tissues allowing the tumor cells to invade these tissues. Eventually, the tumor cells enter the blood or lymphatic system where they repeat the adhesion and invasion steps at a distant (metastatic) site. At this remote site, tumor cells induce the formation of new blood vessels (a process called neovascularization), in and around the growing tumor. These new blood vessels supply nutrients to the metastatic tumor and allow it to grow. Treatments that block any of these steps should act to inhibit metastasis.
Integrins are heterodimers composed of alpha and beta submits that are non-covalently associated. Interactions between integrins and ECM proteins have been shown to be mediated via an Arg-Gly-Asp (RGD) sequence present in the matrix proteins. Both the alpha and beta subunits of the integrin are required for fibrinogen binding.
A well known inhibitor of the integrin-ECM interaction is a disintegrin which represents a family of proteins that include those from venom of snakes of the Crotalidae and Viperidae families have been found to inhibit glycoprotein (GP) IIb/IIIa mediated platelet aggregation. See, e.g., Huang, T. F. et al., J. Biol. Chem. 262:16157 (1987); Gan, Z. R. et al., J. Biol. Chem. 263:19827 (1988); Yasuda, T. et al., J. Am. Coll. Cardiol. 16:714 (1990); Trikha, M. et al., Fibtinolysis 4 (Suppl. 1):105 (1990); Trikha, M. et al., Blood 76 (Suppl. 1):479a (1990); Holahan, M. A. et al., Pharmacology 42:340 (1991); Shebuski, R. J. et al., Circulation 82:169 (1990); Yasuda, T. et al., Circulation 83:1038 (1991). Disintegrins are disulfide rich and, with the exception of barbourin, contain an RGD (Arg-Gly-Asp) sequence that has been implicated in the inhibition of integrin-mediated interactions (Scarborough et al., J. Biol. Chem. 266(20):9359-62 (1991)). Most disintegrins can disrupt different integrin-ECM interactions (e.g., inhibition of β1 integrins (McLane et al. 1998) and β3 integrins such as barbourin are relatively specific and disrupt only αIIbβ3 integrin function (Scarborough et al. (1991)).
The RGD sequence of disintegrins is located at the tip of a flexible loop, the integrin-binding loop, stabilized by disulfide bonds and protruding from the main body of the polypeptide chain. See, e.g., amino acid residues 457 to 469 of SEQ ID NO: 1. This exposed RGD sequence enables disintegrins to bind to integrins with high affinity. Portions of a disintegrin other than the RGD site may have biological effects on integrins. See, e.g., Connolly, T. M. et al., Circulation 82 (Suppl. III):660 (1990)).
Disintegrins that are known to disrupt integrin interactions include bitistatin, an 83 amino acid disintegrin isolated from the venom of Bitis arietans; echistatin, a 49 amino acid disintegrin isolated from the venom of Echis cannatus; kistrin, a 68 amino acid disintegrin isolated from the venom of Calloselasma rhodostoma; trigamin, a 72 amino acid disintegrin isolated from the venom of Trimeresurus gramineus, (see U.S. Pat. No. 5,066,592 by Huang et al.); applaggin, isolated from the venom of Agkistrodon piscivorus piscivorus (see e.g., Chao, B. H. et al., Proc. Natl. Acad. Sci. USA 86:8050 (1989); Savage, B. et al., J. Biol. Chem. 265:11766 (1990)); and contortrostatin (CN), isolated from the venom of Agkistrodon contortix contortix (the southern copperhead snake).
Unlike other monomeric disintegrins, CN is a homodimer with molecular mass (Mr) of 13,505 for the intact molecule and 6,750 for the reduced chains as shown by mass spectrometry (Trikha, Rote, et al., Thrombosis Research 73:39-52 (1994)). CN can be purified from snake venom, as described in Trikha, Rote, et al., Thrombosis Research 73:39-52 (1994).
CN full-length DNA precursor has been cloned and sequenced (Zhou, Hu et al. (2000)). CN is produced in the snake venom gland as a multidomain precursor of 2027 bp having a 1449 bp open reading frame encoding a precursor that includes a pro-protein domain (amino acid residues 1 to 190 of SEQ ID NO: 1), a metalloproteinase domain (residues 191 to 410 of SEQ ID NO: 1) and a disintegrin domain (residues 419 to 483 of SEQ ID NO: 1). The CN precursor is proteolytically processed, possibly autocatalytically, to generate mature CN. The CN disintegrin domain encodes 65 amino acids with a molecular weight equal to that of the mature CN subunit. CN displays the classical RGD motif in its integrin-binding loop.
The CN full-length precursor mRNA sequence can be accessed in the GenBank database using accession number: AF212305. The nucleotide sequence encoding the 65 amino acid disintegrin domain of CN represents the segment from 1339 to 1533 in the mRNA. Plasmids encoding the CN full-length gene have been described (Zhou, Hu et al. (2000)) and are available from the laboratory of Francis S. Markland at University of Southern California (Los Angeles, Calif.). Various recombinant forms of CN are disclosed in U.S. Pat. No. 6,710,030 by Markland.
CN is cysteine-rich (10 cysteines per monomer), displays no secondary structure and, like other disintegrins, has a complex folding pattern that relies on multiple disulfide bonds (four intrachain and two interchain disulfide bonds) to stabilize its tertiary structure (Zhou, Hu et al. (2000)). The compact structure of CN, achieved by its multiple disulfide bonds, renders it more resistant to proteolytic inactivation as compared to other disintegrins.
Receptors of CN that have been identified include: integrins αIIbβ3, αvβ3, αvβ5, and α5β1 (Trikha, De Clerck et al., Cancer Res. 54(18): 4993-98 (1994); Trikha, Rote et al., Thrombosis Res. 73(1): 39-52 (1994); Zhou, Nakada et al., Angiogenesis 3(3): 259-69 (1999); Zhou, Nakada et al., Biochem. Biophys. Res. Commun. 267(1): 350-55 (2000). Interactions between CN and integrins are RGD-dependent. As an anti-cancer agent, CN has effective anti-angiogenic and anti-metastatic properties (Trikha, De Clerck et al. 1994; Trikha, Rote et al. (1994); Schmitmeier et al., Anticancer Res. 20(6B): 4227-33 (2000); Zhou, Hu et al., Biochem. Biophys. 375(2): 278-88 (2000); Markland et al., Haemostasis 31(3-6): 183-91 (2001); Swenson et al., Mol. Cancer Ther. 3(4): 499-511 (2004)). CN also has the ability to directly engage tumor cells and suppress their growth in a cytostatic manner (Trikha, De Clerck et al. (1994); Trikha, Rote et al. (1994); Schmitmeier et al. (2000)). The antitumoral activity of CN is based on its high affinity interaction with integrins α5β1, αvβ3 and αvβ5 on both cancer cells and newly growing vascular endothelial cells (Trikha, De Clerck et al. (1994); Zhou, Nakada et al. (1999); Zhou, Nakada et al. (2000); Zhou, Sherwin et al., Breast Cancer Res. Treat. 61(3): 249-60 (2000)). This diverse mechanism of action provides CN with a distinct advantage over many antiangiogenic agents that only block a single angiogenic pathway and/or do not directly target tumor cells.
The taxanes represent a class of small molecule diterpenoids compounds (i.e., taxoids) that are useful for cancer therapy. Paclitaxel (Taxol®) and docetaxel (Taxotere®), are well known taxanes which are efficacious against a range of solid tumors, particularly carcinomas, melanomas, and sarcomas. (See e.g., references cited in Pamela et al., Clin Cancer Res Vol. 8, 846-855 (2002)). Paclitaxel and docetaxel bind to β tubulin and disrupt microtubule assembly/disassembly. Id. Stabilization of microtubules by taxanes causes mitotic arrest and cell death (e.g., apoptosis) reportedly independent of the p53 tumor suppressor. Id. Taxanes induce genes encoding inflammatory mediators such as tumor necrosis factor alpha, interleukins, and enzymes such as NO synthase and COX-2. Id.
Taxanes have a common “taxoid” core structure shown below.

Taxol® was first isolated from the bark of the Pacific yew (Taxus brevifolia Nutt.) but is presently derived mainly by semisynthesis from the advanced taxoid 10-deacetylbaccatin III, which can be obtained from bark or needles of the European yew, Taxus baccata. (See e.g., references 15-20 in Jennewein, et al., PNAS, 98(24):13595-13560 (2001); see also Holton, et al., J. Am. Chem. Soc., 116:1597-1601 (1994)).
A number of modified taxanes or taxoid analogs have been prepared which have a taxane ring bearing modified side chains. These modified taxanes or taxoid analogs inhibit cancer growth while having greater water solubility and stability than naturally occurring Taxol®. Analogs also include fatty acid conjugates. Exemplary derivatives of Taxol® are described in U.S. Pat. Nos. 6,638,742; 5,278,324; 5,272,171; 5,254,580; 5,250,683; 5,248,796; and 5,227,400; and U.S. Pub. App. No. 2005/0148657; and the references cited therein, as well as those compounds disclosed in Villalva-Servín, et al., Can. J. Chem., 82: 227-39 (2004); Shen, et al., Chem. Pharm. Bull., 53(7): 808-10 (2005); Ono, et al., Biol. Pharm. Bull., 27(3): 345-51 (2004); Sampath, et al., Mol. Cancer Ther., 2(9): 873-74 (2003); and Wolff, et al., Clin. Cancer Res., 9(10): 3589-97 (2003).
The co-administration of taxanes or taxane derivatives with at least one active agent has been reported. Taxotere® in combination with prednisone has been approved by the US Food and Drug Administration for the treatment of metastatic androgen-independent prostate cancer. Rose et al. reported the administration of the oral taxane BMS-275183 in combination with cetuximab (an anti-epidermal growth factor receptor monoclonal antibody) (Rose, et al., Clin. Cancer Res., 10(21): 7413-17 (2004)). Levy, et al. reported the administration of antimetabolite-taxane combinations (specifically, the administration of gemcitabine and docetaxel) in women with anthracycline pretreated metastatic breast cancer (Levy, et al., Cancer Treat. Rev., 31: S17-22 (2005)).